Impaired activation of glycogen synthase in insulin-resistant subjects is associated with both chronically lower activity of protein phosphatase 1 (PP1) and a smaller magnitude of activation of PP1 in response to insulin compared to that seen in insulin-sensitive subjects. Although PP1 activity is low in muscle of resistant subjects, the concentration of catalytic subunit of PP1 (PP1-C), as determined by Western blotting, is higher than in sensitive subjects (B. Nyomba and D. Mott, CDNS, PECRB). Because both of these abnormal characteristics of PP1 in insulin resistance could result from a mutation in the gene for PP1-C, we have begun to examine PP1-C cDNA sequences in muscle of sensitive and resistant subjects and to test the influence of insulin on the concentration of PP1-C RNA in muscle. PP1-C cDNA was amplified by polymerase chain reaction (PCR), ligated with an appropriate vector, cloned and sequenced. Five PCR clones from muscle of an insulin- sensitive subject are virtually identical to the human liver PP1-C cDNA sequence from +138 to +558, except for a base pair change (AC) at nucleotide +237. Several of these clones have an additional base pair change, but since these occur randomly throughout the sequence, they probably arise from the infidelity of Taq polymerase. Three PCR clones of PP1-C from muscle of an insulin-resistant subject have also been sequenced. Two clones are identical to the sequence obtained from muscle of an insulin-sensitive subject. The third clone differs by a 2bp deletion. The concentration of PP1-C RNA was assayed using a 32p-labeled oligonucleotides probe in an S1-nuclease protection assay. The concentration of RNA corresponding to PP1-C in human skeletal muscle decreased by 50% after 30 min of a high dose infusion of insulin in vivo. The concentration returned to basal levels by 120 min.